Construction And Application Of The Transformation System Of Bacillus Subtilis

There is a two-branched innate immune system in plant-microbe interactions. In the first branch innate immune system, plants can recognize and respond to many microbes, including non-pathogens, because of slowly evolving molecules common to many classes of microbes, such as flagellin and EF-Tu and chitin, or other PAMPs. Perception of PAMPs trigger the first branch plant innate immunity and is referred to as PTI. In the second branch innate immune system, plants can recognize pathogen virulence factors, either directly or indirectly. Pathogen virulence effectors from diverse kingdoms are recognized by NB-LRR proteins encoded by most R genes. Perception of pathogen virulence factors triggers the second branch plant innate immunity and is referred to as ETI.The Gram-positive bacterium B. subtilis was considered as a generally recognized as safe organism (GRAS). B. subtilis has stable expression systems, for this reason, the use of B. subtilis for the production of food products and pharmaceutical products are highly favored.In this study, recombinant expression plasmids were transformed into B. subtilis to secrete some plant peptides or pathogen virulence factors. These transformed B. subtilis may be used in agricultural industry. Major results are listed below.1.Construction of the recombinant expression plasmid pHR. In this study, we created the expression pHR vector by combining the following parts of P43promoter, nprB signal peptide, rrnBTlT2terminator, Kanr cassette, amyE5’and3’ parts of B. subtilis168a-amylase gene, and we got the pHR transformed B. subtilis;2. We cloned the ZmPROPEPl gene from maize B73and constructed the ZmPROPEPl gene to plant expression plasmid pCAMBIA3301;3. We synthesized the avr9gene of Cladosporium fulvum, analyzed the structure of an avr4cassette on an agrobacterium-mediated transient expression vector. We constructed the avr9and avr4gene into B. subtilis expression plasmid in many styles, and got many transformed B. subtilis of the two genes; 4.We constructed the B. subtilis expression plasmid pBE2N-avr4and pHY300-avr4. We tested the production of the desired gene of pBE2N-EGFP transformed B. subtilis, the transformation efficiency of two plasmids in different size, and the stability of different plasmids.